{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T13:23:36Z","timestamp":1788269016290,"version":"build-2803163510"},"reference-count":42,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Clostridioides difficile<\/jats:italic>\n (\n C. difficile<\/jats:italic>\n ) is the primary pathogen responsible for nosocomial infectious diarrhea and pseudomembranous colitis. In China, metronidazole and vancomycin are the preferred treatments for\n C. difficile<\/jats:italic>\n infection (CDI). This study aimed to investigate the evolution of vancomycin (VA) and metronidazole (MTZ) resistance, as well as the longitudinal changes in virulence over time, using next-generation sequencing, drug susceptibility tests, and analysis of resistance and virulence genes. Additionally, we monitored the emergence of the highly virulent\n C. difficile<\/jats:italic>\n strain RT027 and the spread and potential outbreak of\n C. difficile<\/jats:italic>\n in the hospital setting. A random stratified sampling method was used to select 114 fecal samples from inpatients at Affiliated Hangzhou First People\u2019s Hospital, School of Medicine, Westlake University, between 2021 and 2024. Clinical data from the enrolled patients were also collected. We conducted antigen and toxin protein detection for\n C. difficile<\/jats:italic>\n , strain isolation and identification, drug sensitivity tests, whole genome sequencing, and bioinformatics analysis. This included comparisons of drug resistance genes, detection of toxin genes, and the construction of phylogenetic trees based on pan-genome analysis to investigate the resistance and toxin gene variations in\n C. difficile<\/jats:italic>\n . Among the 114 samples collected from Affiliated Hangzhou First People\u2019s Hospital, School of Medicine, Westlake University, no vancomycin- or metronidazole-resistant strains were identified. However, the average minimum inhibitory concentration (MIC) of\n C. difficile<\/jats:italic>\n to vancomycin increased annually (\n H<\/jats:italic>\n = 33.208,\n P<\/jats:italic>\n < 0.05). The average MIC of\n C. difficile<\/jats:italic>\n to metronidazole was highest in 2022 but decreased in 2023 and 2024 (\n H<\/jats:italic>\n = 41.990,\n P<\/jats:italic>\n < 0.05). Notably, in 2024, one\n C. difficile<\/jats:italic>\n strain exhibited an MIC for metronidazole at the resistance threshold (2.00 \u03bcg\/mL). Further Spearman correlation analysis of the strain years with drug sensitivity results revealed a positive correlation between strain years and the MIC levels of vancomycin and metronidazole (\n r<\/jats:italic>\n = 0.528,\n P<\/jats:italic>\n < 0.05;\n r<\/jats:italic>\n = 0.377,\n P<\/jats:italic>\n < 0.05). The proportion of toxin-producing strains increased annually, with 100% of strains in 2024 producing toxins, representing the highest proportion compared to the previous three years (\n X<\/jats:italic>\n \u00b2 =11.75,\n P<\/jats:italic>\n < 0.05). Both vancomycin and metronidazole remain effective for the treatment of CDI in clinical practice. However, the sensitivity of\n C. difficile<\/jats:italic>\n to these two drugs is gradually decreasing, and the rate of toxin gene carriage is also rising in clinical cases. No hospital outbreaks of\n C. difficile<\/jats:italic>\n infections were identified in this study.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Clostridioides difficile<\/jats:italic>\n has developed resistance to multiple antibiotics, including cephalosporins, clindamycin, and fluoroquinolones. This has exacerbated the global antibiotic resistance crisis. In China, according to current treatment guidelines, vancomycin and metronidazole are the preferred first-line drugs for treating\n C. difficile<\/jats:italic>\n infections. However, there are reports indicating the emergence of new resistance to both vancomycin and metronidazole. Although there is extensive research on the long-term antibiotic resistance of\n C. difficile<\/jats:italic>\n abroad, research on the continuous monitoring of antibiotic resistance and potential outbreaks of\n C. difficile<\/jats:italic>\n in China is relatively limited. To fill this gap, we studied positive\n C. difficile<\/jats:italic>\n strains from a tertiary general hospital in China. Through Next-Generation Sequencing (NGS), drug sensitivity testing, and analysis of drug resistance and virulence genes, we revealed the evolution of\n C. difficile's<\/jats:italic>\n resistance to vancomycin and metronidazole, as well as changes in virulence, and monitored the spread within the hospital and potential outbreaks of\n C. difficile<\/jats:italic>\n .\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/spectrum.00494-26","type":"journal-article","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T13:00:45Z","timestamp":1784811645000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Longitudinal surveillance of antibiotic resistance and virulence evolution in\n Clostridioides difficile<\/i>\n : a 4-year retrospective study of hospitalized patients in a tertiary hospital in China"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-7393-767X","authenticated-orcid":false,"given":"Junjie","family":"Lao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05hfa4n20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Laboratory, Affiliated Hangzhou First People\u2019s Hospital, School of Medicine, Westlake University","place":["Hangzhou, China"]},{"id":[{"id":"https:\/\/ror.org\/04epb4p87","id-type":"ROR","asserted-by":"publisher"}],"name":"The Fourth School of Clinical Medicine, Hangzhou First People\u2019s Hospital, Zhejiang Chinese Medical University","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingli","family":"Zhang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04epb4p87","id-type":"ROR","asserted-by":"publisher"}],"name":"The Fourth School of Clinical Medicine, Hangzhou First People\u2019s Hospital, Zhejiang Chinese Medical University","place":["Hangzhou, China"]},{"id":[{"id":"https:\/\/ror.org\/00a2xv884","id-type":"ROR","asserted-by":"publisher"}],"name":"The First Affiliated Hospital, Zhejiang University School of Medicine, Liangzhu Branch","place":["Hangzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinghan","family":"Huang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05hfa4n20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Laboratory, Affiliated Hangzhou First People\u2019s Hospital, School of Medicine, Westlake University","place":["Hangzhou, China"]},{"id":[{"id":"https:\/\/ror.org\/04epb4p87","id-type":"ROR","asserted-by":"publisher"}],"name":"The Fourth School of Clinical Medicine, Hangzhou First People\u2019s Hospital, Zhejiang Chinese Medical University","place":["Hangzhou, 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